The specific gravity of an aqueous test sample, such as urine or serum, is a measure of the relative proportions of solid materials dissolved in the test sample to the total volume of the test sample. In general, the specific gravity of an aqueous test sample is a measure of the relative degree of concentration or the relative degree of dilution of the test sample. In regard to urine samples, the assay for specific gravity is important to help interpret the results of the other assays performed in a routine urinalysis. Clinically, under appropriate and standardized conditions of fluid restriction or increased fluid intake, the specific gravity of a urine sample measures the concentrating and diluting abilities of the kidneys of an individual.
Normally, the specific gravity of urine ranges from about 1.005 to about 1.030, but most often urine specific gravity ranges from about 1.010 to about 1.025. The specific gravity of urine is highest in the first morning urine specimen and generally is greater than 1.020. A specific gravity of about 1.025 or above in a random first morning urine specimen indicates a normal concentrating ability of the kidneys. The ability of the kidneys to concentrate urine can be measured by a concentration test. The concentration test is performed by withholding all fluids from the individual after the evening meal. Then the urine excreted during the night is discarded, and the first morning specimen is assayed. Clinically, a urine specific gravity of 1.025 or higher is considered normal, and indicates a normally functioning kidney.
Dilution tests also are used to determine the ability of the kidneys to concentrate liquids. However, these tests are less useful than the concentration tests because dilution tests provide less information about renal functions. Furthermore, dilution tests are potentially hazardous to the patient. For example, patients afflicted with certain diseases, such as Addison's disease, are advised to avoid dilution tests. The dilution test requires the patient to drink a suitable water load, usually about one liter during a 30-minute period. Then, within approximately one hour, normal patients will excrete at least one urine specimen with a specific gravity less than 1.003.
Either an abnormally low urine specific gravity or an abnormally high urine specific gravity is clinically significant. For example, diabetes insipidus, a disease caused by the absence of, or impairment to, the normal functioning of the antidiuretic hormone (ADH), is the most severe example of impaired kidney concentrating ability. This disease is characterized by excreting large urine volumes of low specific gravity. The urine specific gravity of individuals suffering diabetes insipidus usually ranges between 1.001 and 1.003. Low urine specific gravity also occurs in persons suffering from glomerulonephritis, pyelonephritis, and various other renal anomalies. In these cases, the kidney has lost its ability to concentrate the urine because of tubular damage.
An abnormally high urine specific gravity also is indicative of a diseased state. For example, the urine specific gravity is high in persons suffering from diabetes mellitus, adrenal insufficiency, hepatic disease, or congestive cardiac failure. The specific gravity likewise is elevated whenever there has been excessive loss of water, such as with sweating, fever, vomiting, and diarrhea. In addition, abnormally high amounts of certain urinary constitutents, in particular glucose and protein, increase the urine specific gravity of some individuals suffering from diabetes mellitus or nephrosis up to 1.050 or greater. As a general rule, the specific gravity increases 0.004 for every 1% glucose in urine and 0.003 for every 1% protein in urine. Furthermore, urine with a fixed low specific gravity of approximately 1.010 that varies little from specimen to specimen is known as isothenuric. This condition is indicative of severe renal damage with disturbance of both the concentrating and diluting abilities of the kidney.
Therefore, in order to determine if an individual consistently has either an abnormally high or an abnormally low urine specific gravity, and in order to help monitor the course of a medical treatment to determine its effectiveness, simple, accurate and inexpensive specific gravity assays have been developed. In general, the specific gravity of a test sample is a measurement that relates to the density of the test sample. The specific gravity is a value derived from the ratio of the weight of a given volume of a test sample, such as urine, to the weight of the same volume of water under standardized conditions (Eq. 1). ##EQU1## Water has a specific gravity of 1.000. Since urine is a solution of minerals, salts, and organic compounds in water, the specific gravity of urine is greater than 1.000. The relative difference reflects the degree of concentration of the urine specimen and is a measure of the total solids in urine.
Several methods are available to determine the specific gravity of urine. The most widely used method, and possibly the least accurate, employs a urinometer. The urinometer is a weighted, bulb-shaped instrument having a cylindrical stem containing a scale calibrated in specific gravity readings. The urinometer is floated in a cylinder containing the urine sample, and the specific gravity of the urine is determined by the depth the urinometer sinks in the urine sample. The specific gravity value is read directly from the urinometer scale at the junction of the urine with the air. The urinometer method is cumbersome and suffers from the disadvantages of requiring large volumes of urine test sample, difficult and inaccurate reading of the urinometer scale and unreliable assays because the urinometer is not regularly recalibrated. In addition, each urinometer is calibrated to read 1.000 in distilled water at a specific temperature indicated on each instrument. There is a change in the specific gravity of 0.001 for each 3.degree. C. above and below this temperature. Therefore, for precise work, temperature corrections must be made on the readings. Corrections also are recommended when glucose or protein is present in the urine sample.
Refractometry provides an indirect method of measuring the specific gravity of urine. The refractive index is the ratio of the velocity of light in air to the velocity of light in solution. The refractive index is not identical to the specific gravity of urine, but the refractive index can be correlated to the specific gravity. The refractive index of urine varies directly with the number of dissolved particles in urine and, therefore, varies directly with the specific gravity of urine. Consequently, measurement of the refractive index of urine can be related to the specific gravity of urine.
The refractometer method of determining the specific gravity of urine is desirable because specific gravity measurements are possible on as little as one drop of urine. The refractometer used to determine the refractive index is a small hand held instrument calibrated in terms of specific gravity, refractive index and total solid content. The refractometer requires a drop of urine placed in the appropriate sample slot in the refractometer. The instrument is held towards a light source and the assay, either in terms of specific gravity, refractive index, or solid content, is read directly from the calibrated scale located in the eyepiece. The specific gravity scale on the refractometer reads from 1.000 to 1.035 in increments of 0.001. The refractometer has the disadvantage of requiring daily calibration and not being amenable to home assays.
A third urinalysis method for specific gravity, the falling drop method, like the urinometer, is a direct measurement of urine specific gravity. In accordance with this method, a drop of urine is introduced into each of a series of columns that are filled with solvent mixtures of increasing and known specific gravity. When the drop of urine comes to rest after its initial momentum has dissipated and then neither rises nor falls, the specific gravity of the urine is determined to be the same as the solvent mixture of that particular column. In this procedure, a series of mixtures of xylene and bromobenzene, chloroform and benzene, or bromobenzene and kerosene have been employed. Prior to development of the refractometer, this technique had the advantage of requiring only a few drops of test sample to conduct a specific gravity assay. The falling drop method, however, never achieved widespread use in routine urinalysis because of the obvious time requirements in setting up such a system and the inability for an individual to perform the assay at home.
The falling drop method described above also can be performed instrumentally. Unlike the graded series of solvent mixtures described above, the instrument-based assay uses a specially designed column filled with a silicone oil having a controlled specific gravity and viscosity. The column is designed to measure the time required for a precisely measured drop of test sample to fall a distance defined by two optical gates (lamp-phototransistor pairs) mounted one above the other in a temperature-controlled column filled with a water-immiscible silicone oil of a slightly lower density than the test sample. The light beams from the lamps travel through the column oil and strike phototransistors located on the opposite wall of the column. A drop of urine dispensed into the column oil by a pipette breaks the beams of light as it falls through the oil. The urine drop breaking the upper beam starts an electronic timer, and breaking the lower beam stops the timer. The falling time is measured electronically and computed into specific gravity units. This specific gravity method is very precise, however, the cost of the assay instrument and the degree of skill required to operate the instrument makes home testing for urine specific gravity impractical.
Each of the above described instrument-based specific gravity assay methods has disadvantages, whereby none of the assay methods are particularly well suited to performing specific gravity assays outside of the physician's office or laboratory. Consequently, reagent impregnated test strips have been developed to allow specific gravity assays to be performed at home. The test strip assay developed for specific gravity measurements is an indirect assay method, wherein the test strip changes color in response to the ionic strength of the urine sample.
The present day specific gravity test strips comprise a carrier matrix impregnated with a reagent composition including three essential ingredients: a polyelectrolyte, such as a partially neutralized poly(methyl vinyl ether/maleic acid); a chromogenic indicator, such as bromothymol blue; and suitable buffering agents. This reagent composition is sensitive to the number of ions, or electrolytes, in the urine sample, such that the polyelectrolyte of the reagent composition undergoes a pKa (acid dissociation constant) change in relation to the ionic strength of the urine sample. Therefore, as the concentration of electrolytes in the urine increases (high specific gravity), the pKa of the polyelectrolyte present in the reagent composition decreases because free carboxyl groups are converted to carboxylate groups. The overall result is a pH decrease and a color transition of the bromothymol blue chromogenic indicator from blue-green to green to yellow-green in response to increased specific gravities. The resulting color transition, indicating a pH change caused by increasing ionic strength, or increasing specific gravity, is empirically related to the specific gravity of the test sample.
Some test strips used in specific gravity assays have a single test area consisting of a small square pad of a carrier matrix impregnated with the buffered polyelectrolyte and chromogenic indicator dye composition. Other test strips are multideterminant reagent strips that include one test area for the specific gravity assay as described above, and further include several additional test areas on the same strip to permit the simultaneous assay of other urinary constituents. For both types of reagent impregnated test strips, the assay for the specific gravity of urine is performed simply by dipping the test strip into a well mixed, uncentrifuged urine sample, then comparing the resulting color of the test area of the test strip to a standardized color chart provided on the test strip bottle.
For test strips utilizing the partially neutralized poly(methyl vinyl ether/maleic acid) polyelectrolyte and bromothymol blue indicator, semiquantitative assays for the specific gravity of aqueous test samples can be performed and reported as specific gravities ranging from 1.000 to 1.030. A reading of 1.000, or a blue-green color, indicates that the urine has a very low specific gravity, as demonstrated by the lack of a color transition of the chromogenic indicator dye. A specific gravity reading of from 1.005 to 1.030 is signified by color transitions, of from blue-green through green to yellow-green, that serve as reliable indicators of increasing specific gravity.
In accordance with the present day reagent strip method, an individual can readily determine, visually, that the specific gravity of a urine sample is in the range of about 1.000 to about 1.030. However, the sensitivity and the color resolution afforded by the presently available commercial test strips is insufficient to permit differentiation between liquid test samples having different, but nearly identical, specific gravities, such as specific gravities that differ by 0.003. The inability to differentiate between test samples having different, but nearly identical, specific gravities is important clinically because a healthy person usually has a urine specific gravity in the range of about 1.005 to about 1.030. Therefore, it could be important to more precisely determine a urine specific gravity that is either slightly above or slightly below these normal values, such that the accurate specific gravity assay can be interpreted in conjunction with assays for other urine analytes to provide a reliable diagnosis and to allow correct medical treatment to be instituted.
Therefore, it would be extremely advantageous to have a simple, accurate and trustworthy method of assaying for urine specific gravity that allows visual differentiation of specific gravity values within the ranges of 1.000 to about 1.005, about 1.005 to about 1.010, and about 1.010 to about 1.015, and upwards to between about 1.045 to about 1.050. By providing an accurate method of determining urine specific gravity in an easy to use form, such as a dip-and-read test strip, the urine assay can be performed by laboratory personnel to afford immediate test results. The specific gravity assay results can be interpreted in conjunction with assays for other urine constituents, such that a diagnosis can be made without having to wait for assay results and medical treatment can be commenced immediately. Furthermore, the test strip method can be performed by the patient at home to more precisely determine the specific gravity of the urine and therefore to help monitor the success of the medical treatment the patient is undergoing.
As will be described more fully hereinafter, the method of the present invention allows the fast, accurate and trustworthy assay for the specific gravity of urine by utilizing a test strip that includes a specific gravity reagent composition incorporating a molybdate-dye complex. The specific gravity reagent composition including the molybdate-dye complex improves the sensitivity of the assay and provides sufficient visual color differentiation between urine samples having specific gravities differing by as little as 0.003 in the specific gravity range of approximately 1.000 to approximately 1.030, and between urine samples having specific gravities differing by as little as 0.005 from approximately 1.030 to approximately 1.050. Therefore urine specific gravities of from approximately 1.000 to approximately 1.050 can be accurately determined.
The urine specific gravity of an individual depends upon the precise nature of his pathological disorder and upon the severity of his specific disease. An abnormally high or abnormally low urine specific gravity can be intermittent or continuous. Therefore, accurate and reliable specific gravity assays of urine and other aqueous test samples must be available for both laboratory and home use. The assays must permit the accurate measurement of abnormally low and abnormally high specific gravities, such that a correct diagnosis can be made and correct medical treatment implemented, monitored and maintained. In addition, it would be advantageous if the specific gravity assay method could be utilized in a dip-and-read format for the easy and economical determination of urine or other aqueous test sample specific gravities.
Furthermore, any method of assaying for the specific gravity of urine or other aqueous test samples must yield accurate, trustworthy and reproducible results by utilizing a specific gravity reagent composition that undergoes a color transition as a result of an interaction in response to the specific gravity of the test sample, and not as a result of a competing chemical or physical interaction, such as a pH change or preferential interaction with another test sample component, like protein or glucose. Moreover, it would be advantageous if the specific gravity assay method utilizing dry reagent strips provides for the rapid, economical and accurate determination of urine or other aqueous test sample specific gravities. Additionally, the method and composition utilized in the specific gravity assay should not adversely affect or interfere with the other test reagent pads that are present on multiple test pad strips.
Prior to the present invention, no known method of assaying urine or other aqueous test samples for specific gravity included a reagent composition providing sufficient sensitivity and color differentiation to allow accurate and trustworthy specific gravity assays to be made in the range of from about 1.000 to about 1.050. In addition, although a dry phase reagent test strip utilizing a partially neutralized polyelectrolyte and a dye, such as bromothymol blue, has been used extensively, no dry phase test strip has incorporated a molybdate-dye complex to provide sufficient sensitivity and sufficient visual color resolution to allow specific gravity differentiation between test samples having specific gravities differing by as little as 0.003.
The prior art contains numerous references to the polyelectrolyte-dye chemistry utilized in the specific gravity assay of urine. For example, U.S. Pat. Nos. 4,318,709 and 4,376,827 disclose the basic polyelectrolyte-dye technique used to assay for urine specific gravity. Both patents teach utilizing polyelectrolyte-dye chemistry to determine the specific gravity of urine by monitoring the color transition of the dye.
However, as will be fully described in the detailed description of the invention, the present invention provides a composition and method for the accurate determination of urine and other aqueous test sample specific gravities by utilizing a molybdate-dye complex as the indicator component of a specific gravity reagent composition. The molybdate-dye complex is known to interact with proteins in a test sample to produce a color transition. However, in accordance with an important feature of the present invention, it has been found that the color transition resulting from the interaction between the molybdate-dye complex and proteins is very sensitive to the ionic strength of the test sample, and therefore also is sensitive to the specific gravity of the test sample. As a result, it has been demonstrated that the sensitivity of the molybdate-dye complex and protein interaction to test sample ionic strength, or specific gravity, provides an indirect, but accurate, method of determining aqueous test sample specific gravities.
The publication "Color Reaction Between Pyrogallol Red-Molybdenum (VI) Complex and Protein", Y. Fujita, I. Mori, and S. Kitano, Bunseki Kagaku, 32, pp. E379-E386 (1983), first described the interaction between a protein and a pyrogallol red-molybdenum complex. The reported method required the incorporation of a chelating agent or metal ion into the molybdate-dye complex in order to determine the protein concentration of a test sample.
Similarly, Japanese Patent No. 61/155757 (1986) disclosed a colorimetric method of assaying for proteins in a test sample by using a composition including a molybdenum-dye complex and either a chelating agent or certain metal ions. However, it has been found that the method disclosed in Japanese Patent No. 61/155757 suffers from a severe ionic strength, or specific gravity, interference. It has been demonstrated that the degree of molybdate-dye complex binding to the protein, and therefore the degree of color transition, is inversely related to the ionic strength of the sample. As a result, the assay of a urine sample of low ionic strength (low specific gravity) produces a greater color transition in the test device (therefore indicating a greater protein content) than the assay of a urine sample having the same protein content, but a higher ionic strength (higher specific gravity). Unexpectedly, the specific gravity reagent composition utilized in the present invention takes advantage of the ionic strength/specific gravity interference found in the protein assays to provide accurate specific gravity assays regardless of other test sample components, such as proteins
The publication, "Urinary Protein as Measured with a Pyrogallol Red-Molybdate Complex, Manually and in a Hitachi 726 Automated Analyzer", N. Watanabe, S. Kamei, A. Ohkubo, M. Yamanaka, S. Ohsawa, K. Makino and K. Tokuda, Clin. Chem., 32/8, pages 1551-1554 (1986), further describes the method disclosed in Japanese Patent No. 61/155757. The Watanabe publication describes the automated or manual detection of proteins in urine using a molybdate-dye complex. The publication reports that the interaction of interest between the protein and the molybdate-dye complex continued for at least eight minutes and is complete within 10 minutes at 37.degree. C. for automated assays, but for manual assays, the interaction was allowed to continue for 20 minutes before the assay was examined for a response. In addition, to the ionic strength interference described above, such a long interaction time for the complete color transition to occur is both inconvenient and can lead to erroneous assays should the degree of color transition, and hence protein content, be determined too quickly. However, according to the method of the present invention, the assay for the specific gravity of a test sample using a specific gravity reagent composition including a molybdate-dye complex is essentially complete in less than two minutes, therefore providing fast specific gravity results with a greatly reduced probability of an erroneous assay.
In contrast to the prior art, and in contrast to the presently available commercial test strips, the method of the present invention provides increased sensitivity in the measurement of urine specific gravity by utilizing a specific gravity reagent composition including a molybdate-dye complex, thereby achieving an accurate specific gravity assay, such as to within 0.003 for liquids having a specific gravity of from about 1.000 to about 1.030, and to within 0.005 for liquids having a specific gravity of from about 1.030 to about 1.050. Unexpectedly and surprisingly, the method of the present invention, also in contrast to the prior art, allows the simple and fast measurement of the specific gravity of a liquid test sample. Hence, in accordance with the method of the present invention, new and unexpected results are achieved in the dry phase reagent strip assay of urine and other aqueous test samples for specific gravity, in the range of from about 1.000 to about 1.050, by utilizing a specific gravity reagent composition including a molybdate-dye complex.